Formation and dimerization of the phosphodiesterase active site of the Pseudomonas aeruginosa MorA, a bi-functional c-di-GMP regulator

Diguanylate cyclases (DGC) and phosphodiesterases (PDE) respectively synthesise and hydrolyse the secondary messenger cyclic dimeric GMP (c-di-GMP), and both activities are often found in a single protein. Intracellular c-di-GMP levels in turn regulate bacterial motility, virulence and biofilm formation. We report the first structure of a tandem DGC–PDE fragment, in which the catalytic domains are shown to be active. Two phosphodiesterase states are distinguished by active site formation. The structures, in the presence or absence of c-di-GMP, suggest that dimerisation and binding pocket formation are linked, with dimerisation being required for catalytic activity. An understanding of PDE activation is important, as biofilm dispersal via c-di-GMP hydrolysis has therapeutic effects on chronic infections

The impact of exchanging the light and heavy chains on the structures of bovine ultralong antibodies

The third complementary‐determining regions of the heavy‐chain (CDR3H) variable regions (VH) of some cattle antibodies are highly extended, consisting of 48 or more residues. These `ultralong' CDR3Hs form β‐ribbon stalks that protrude from the surface of the antibody with a disulfide cross‐linked knob region at their apex that dominates antigen interactions over the other CDR loops. The structure of the Fab fragment of a naturally paired bovine ultralong antibody (D08), identified by single B‐cell sequencing, has been determined to 1.6 Å resolution. By swapping the D08 native light chain with that of an unrelated antigen‐unknown ultralong antibody, it is shown that interactions between the CDR3s of the variable domains potentially affect the fine positioning of the ultralong CDR3H; however, comparison with other crystallographic structures shows that crystalline packing is also a major contributor. It is concluded that, on balance, the exact positioning of ultralong CDR3H loops is most likely to be due to the constraints of crystal packing

Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics

Structural and functional characterization of nanobodies that neutralize Omicron variants of SARS-CoV-2

The Omicron strains of SARS-CoV-2 pose a significant challenge to the development of effective antibody-based treatments as immune evasion has compromised most available immune therapeutics. Therefore, in the ‘arms race’ with the virus, there is a continuing need to identify new biologics for the prevention or treatment of SARS-CoV-2 infections. Here, we report the isolation of nanobodies that bind to the Omicron BA.1 spike protein by screening nanobody phage display libraries previously generated from llamas immunized with either the Wuhan or Beta spike proteins. The structure and binding properties of three of these nanobodies (A8, H6 and B5-5) have been characterized in detail providing insight into their binding epitopes on the Omicron spike protein. Trimeric versions of H6 and B5-5 neutralized the SARS-CoV-2 variant of concern BA.5 both in vitro and in the hamster model of COVID-19 following nasal administration. Thus, either alone or in combination could serve as starting points for the development of new anti-viral immunotherapeutics

Ligand binding to pentraxins

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) have emerged as potentially important targets in the treatment of amyloidosis and cardiovascular diseases respectively, although their normal physiological functions are unclear. Structurally highly conserved homologous proteins are present in common experimental animals such as the rat, mouse, rabbit and hamster but there are major differences from the human p,entraxins in their normal behaviour as acute phase proteins, fine ligand specificity and capacity to activate the complement system. . SAP binds to amyloid fibrils ofall types and may contribute to their formation, stabilisation and persistence. In order to extend our current knowledge ofligand recognition by SAP, the crystal structures ofSAP complexed to two ligands, Methylmalonic acid and Phosphatidylethanolamine, have been solved to 1.6 Aand 1.4Aresolution respectively. Since important biological functions ofproteins are often conserved among species, the structural differences between the rat and human pentraxins were investigated. The crystal structure ofrat SAP was solved to 2.2 Aresolution by molecular replacement. This pentameric structure displayed subtle differences in the electrostatic properties. It remains to be determined whether this has an effect on avid binding of SAP to DNA, a functional property ofh~manSAP still poorly understood. CRP, a pentraxin traditionally defined by its binding affinity for PC, was studied in complex with PE. The crystal structure ofthe CRP-PE complex at 2.7 Aresolution revealed that the nitrogen end ofPE dips further downwards into the hydrophobic pocket ofCRP than PC. CRP-mediated complement activation can exacerbate ischemic tissue injury in the heart as well as in the brain. Therefore, knowledge ofthe exact stoichiometry and the protein-protein interactions between CRP and C1q may aid the development ofsmall molecules capable ofdisrupting such protein-protein interactions. Purification of C1q has been achieved by ion-exchange chromatography and gel filtration from BPL paste. Crystallisation trials have been performed, however no crystals have been observed that contain the protein-protein complex.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Ligand binding to pentraxins

The human pentraxin proteins, serum amyloid P component (SAP) and C-reactive protein (CRP) have emerged as potentially important targets in the treatment of amyloidosis and cardiovascular diseases respectively, although their normal physiological functions are unclear. Structurally highly conserved homologous proteins are present in common experimental animals such as the rat, mouse, rabbit and hamster but there are major differences from the human pentraxins in their normal behaviour as acute phase proteins, fine ligand specificity and capacity to activate the complement system. SAP binds to amyloid fibrils of all types and may contribute to their formation, stabilisation and persistence. In order to extend our current knowledge of ligand recognition by SAP, the crystal structures of SAP complexed to two ligands, Methylmalonic acid and Phosphatidylethanolamine, have been solved to 1.6 A and 1.4 A resolution respectively. Since important biological functions of proteins are often conserved among species, the structural differences between the rat and human pentraxins were investigated. The crystal structure of rat SAP was solved to 2.2 A resolution by molecular replacement. This pentameric structure displayed subtle differences in the electrostatic properties. It remains to be determined whether this has an effect on avid binding of SAP to DNA, a functional property of human SAP still poorly understood. CRP, a pentraxin traditionally defined by its binding affinity for PC, was studied in complex with PE. The crystal structure of the CRP-PE complex at 2.7 A resolution revealed that the nitrogen end of PE dips further downwards into the hydrophobic pocket of CRP than PC. CRP-mediated complement activation can exacerbate ischemic tissue injury in the heart as well as in the brain. Therefore, knowledge of the exact stoichiometry and the protein-protein interactions between CRP and Clq may aid the development of small molecules capable of disrupting such protein-protein interactions. Purification of Clq has been achieved by ion-exchange chromatography and gel filtration from BPL paste. Crystallisation trials have been performed, however no crystals have been observed that contain the protein-protein complex.</p

Characterization of recombinantly expressed matrilin VWA domains

VWA domains are the predominant independent folding units within matrilins and mediate protein–protein interactions. Mutations in the matrilin-3 VWA domain cause various skeletal diseases. The analysis of the pathological mechanisms is hampered by the lack of detailed structural information on matrilin VWA domains. Attempts to resolve their structures were hindered by low solubility and a tendency to aggregation. We therefore took a comprehensive approach to improve the recombinant expression of functional matrilin VWA domains to enable X-ray crystallography and nuclear magnetic resonance (NMR) studies. The focus was on expression in Escherichia coli, as this allows incorporation of isotope-labeled amino acids, and on finding conditions that enhance solubility. Indeed, circular dichroism (CD) and NMR measurements indicated a proper folding of the bacterially expressed domains and, interestingly, expression of zebrafish matrilin VWA domains and addition of N-ethylmaleimide yielded the most stable proteins. However, such proteins did still not crystallize and allowed only partial peak assignment in NMR. Moreover, bacterially expressed matrilin VWA domains differ in their solubility and functional properties from the same domains expressed in eukaryotic cells. Structural studies of matrilin VWA domains will depend on the use of eukaryotic expression systems

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection

Elongation Factor 2 Kinase is regulated by Proline Hydroxylation and protects cells during Hypoxia

Protein synthesis, and especially translation elongation, requires large amounts of energy, which is often generated by oxidative metabolism. Elongation is controlled by phosphorylation of eukaryotic elongation factor 2 (eEF2), which inhibits its activity and is catalysed by eEF2 kinase (eEF2K), a calcium/calmodulin-dependent ?-kinase.Hypoxia causes the activation of eEF2K and induces eEF2 phosphorylation independently of previously-known inputs into eEF2K. Here, we show that eEF2K is subject to hydroxylation on proline-98. Proline hydroxylation is catalysed by proline hydroxylases, oxygen-dependent enzymes which are inactivated during hypoxia. Pharmacological inhibition of proline hydroxylases also stimulates eEF2 phosphorylation. Pro98 lies in a universally-conserved linker between the calmodulin-binding and catalytic domains of eEF2K. Its hydroxylation partially impairs the binding of calmodulin to eEF2K and markedly limits the CaM-stimulated activity of eEF2K. Neuronal cells depend on oxygen and eEF2K helps to protect them from hypoxia.eEF2K is the first example of a protein directly involved in a major energy-consuming process to be regulated by proline hydroxylation. Since eEF2K is cytoprotective during hypoxia and other conditions of nutrient insufficiency, it may be a valuable target for therapy of poorly-vascularised solid tumours